Process for preparing active carbonates of polyalkylene oxides for modification of polypeptides

ABSTRACT

Poly(ethylene glycol)-N-succinimide carbonate and its preparation are disclosed. Polyethylene glycol (PEG) is converted into its N-succinimide carbonate derivative. This form of the polymer reacts readily with amino groups of proteins in aqueous buffers. The modified proteins have PEG-chains grafted onto the polypeptide backbone by means of stable, hydrolysis-resistant urethane (carbamate) linkages.

This application is a division of application Ser. No. 08/198,193 filedFeb. 17, 1994. now U.S. Pat. No. 5,612,460 and in turn is a division ofSer. No. 08/817,757 filed Jan. 8, 1992, U.S. Pat. No. 5,324,844, whichin turn is a division of Ser. No. 07/511,243 filed Apr. 19, 1990 U.S.Pat. No. 5,122,614 which is a continuation-in-part of Ser. No.07/340,928, Apr. 19, 1989, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to chemical modification of polypeptidesby means of covalent attachment of strands of polyalkylene oxide to apolypeptide molecule such as is disclosed in U.S. Pat. No. 4,179,337, toDavis, et al. It is disclosed in Abuchowski & Davis "Enzymes as Drugs",Holcenberg & Roberts, eds., pp. 367-383, John Wiley & Sons, N.Y. (1981)that such preparations of polypeptides have reduced immunogenicity andantigenicity and also have a longer lifetime in the bloodstream ascompared to the parent polypeptides. These beneficial properties of themodified polypeptides make them very useful in a variety of therapeuticapplications, such as enzyme therapy.

The active groups that are introduced onto polyalkylene oxides for thepurpose of subsequent attachment of these polymers to proteins mustsatisfy the following requirements:

1. The active groups have to be reactive enough to afford fast reactionwith a protein under mild conditions;

2. The residues released from the active groups during the process ofmodification have to be non-toxic and/or readily separable from theprotein-polymer adduct.

To effect covalent attachment of polyethylene glycol (PEG) to a protein,the hydroxyl end-groups of the polymer must first be converted intoreactive functional groups. This process is frequently referred to as"activation" and the product is called "activated PEG".Methoxypolyethylene glycol (mPEG) derivatives, capped on one end with afunctional group, reactive towards amines on a protein molecule, areused in most cases.

The most common form of activated PEG heretofore used for preparation oftherapeutic enzymes is poly(ethylene glycol)succinoyl-N-hydroxysuccinimide ester (SS-PEG) Abuchowski, et al. CancerBiochem. Biophys. 7, 175-186 (1984), Scheme 1!. Use of this activatedpolymer satisfies both of the requirements listed above. However, it hasone major drawback. The ester linkage between the polymer and succinicacid residue has limited stability in aqueous media U.S. Pat. No.4,670,417, to Iwasaki, et al. (1987); Ulbrich, et al., Makromol. Chem.187, 1131-1144 (1986)!. ##STR1##

Various functionalized polyethylene glycols (PEG) have been effectivelyused in such fields as protein modification (Abuchowski & Davis, 1981,supra), peptide chemistry Zalipsky, et al., Int. J. Peptide ProteinRes., 30, 740-783 (1987)! and preparation of conjugates withbiologically active materials Zalipsky, et al., Eur. Polym. J. 19,1177-1183 (1983) and Zalipsky and Barany, Polymer Preprints, Am. Chem.Soc. Div. Polym. Chem. 27(1), 1-2 (1986)!. PEG protein conjugates usefulin medical applications have shown promise, particularly with regard totheir stability to proteolytic digestion, reduced immunological responseand longer half-life times in the bloodstream.

To accomplish this, the prior art has activated the hydroxy group of PEGwith cyanuric chloride and the resulting compound then coupled withproteins (Abuchowski, et al. (1977) J. Biol. Chem. 252, 3578; Abuchowskiand Davis, 1981, supra). However, various disadvantages of using thismethod exist, such as the toxicity of cyanuric chloride and thenon-specific reactivity for proteins having functional groups other thanamines, such as free essential cysteine or tyrosine residues.

In order to overcome these and other disadvantages, alternativeprocedures, such as succinimidyl succinate derivatives of PEG (SS-PEG)have been introduced (Abuchowski, et al. 1984, supra, see Scheme 1,above). It reacts quickly with proteins (30 min) under mild conditionsyielding active yet extensively modified conjugates use of thisactivated polymer has one major disadvantage. The ester linkage betweenthe polymer and the succinic acid residue has limited stability inaqueous media U.S. Pat. No. 4,670,417 to Iwasaki, et al. and Ulbrich, etal. Makromol Chem., 187, 1131-1144 (1986)!.

Formation of urethane linkages between amino groups of a protein and PEGovercomes the problem of hydrolytic loss of the polymer chains Veronese,et al., Appl. Biochem. Biotechnol. 11, 141-152 (1985)!. In fact, it wasdemonstrated on radioactively labeled PEG-derivatives that urethanelinks are completely stable under a variety of physiological conditionsLarwood & Szoka J., Labeled Compounds Radiopharm., 21, 603-614 (1984)!.The attachment of PEG to a protein via a carbamate derivative wasaccomplished Beauchamp, et al. Analyt. Biochem. 131, 25-33 (1983)! usingcarbonyldiimidazole-activated PEG. However, the polymer activated inthis manner is not very reactive and therefore long reaction times(48-72 hrs at pH 8.5) were required to achieve sufficient modifications.Therefore, the carbonyldiimidazole-activated agent clearly does notsatisfy the first requirement noted above. An additional disadvantage ofthis approach is in the relatively high cost of carbonyldiimidazole.

Use of PEG-phenylcarbonate derivatives for preparation ofurethane-linked PEG-proteins was reported see Veronese, et al. (1985),supra!. The main drawback of this approach lies in the toxicity of thehydrophobic phenol residues (p-nitrophenol or 2, 4, 5-trichlorophenol)and their affinity for proteins. Clearly this method does not satisfythe second requirement noted above.

Each of the activated forms of the polymer has properties which can beconsidered advantageous or disadvantageous, depending on the system ofuse. In light of the many applications of PEG-polypeptides, it isdesirable to broaden the arsenal of protein modifying PEG-reagents madefor a specific end use.

SUMMARY OF THE INVENTION

The present invention provides a compound having the structure ##STR2##wherein R₁ is H--, H₃ C--, an oxycarbonyl N-dicarboximide group, or anyother functional group;

wherein each R₂, R₃, and R₄ is an alkyl group which may be straight,branched, disubstituted, or unsubstituted, and wherein each R₂, R₃, andR₄ may be independently the same as, or different from, the others ofR₂, R₃, and R₄ ;

wherein R₅ is an N-dicarboximide group; and

wherein a is an integer between 1 and 1000 and each of b and c is aninteger between 0 and 1000, and the sum of a, b, and c is between 10 and1000.

The present invention also provides a process for preparing the compoundwhich comprises reacting a compound having the structure ##STR3##wherein R₁ is H--, H₃ C--, or any other functional group; wherein eachR₂, R₃, and R₄ is an alkyl group which may be straight, branched,disubstituted, or unsubstituted, and wherein each R₂, R₃, and R₄ may beindependently the same as, or different from, the others of R₂, R₃, andR₄ ;

wherein X is a halogen;

wherein a is an integer between 1 and 1000 and each of b and c is aninteger between 0 and 1000, and the sum of a, b, and c is between 10 and1000.

with an N-hydroxydicarboximide in the presence of a base.

The present invention further provides a modified polypeptide comprisinga polypeptide having bound thereto at least one polymer having thestructure ##STR4## wherein R₁ is H--, H₃ C--, onoxycarbonyl-N-dicarboximide or any other functional group;

wherein each R₂, R₃, and R₄ is an alkyl group which may be straight,branched, substituted, or unsubstituted, and wherein each R₂, R₃, and R₄may be independently the same as, or different from, the others of R₂,R₃, and R₄.

wherein a is an integer between 1 and 1000 and each of b and c is aninteger between 0 and 1000, and the sum of a, b, and c is between 10 and1000.

wherein each polymer is covalently bound to an amine group of thepolypeptide by a urethane linkage.

The invention also provides a process for preparing a modifiedpolypeptide comprising reacting a polypeptide with the compound at a pHin the range of about 5.8-11.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Release of mPEG from PEG-BSA conjugates.

Experimental Conditions

Solutions of both types of PEG-BSA conjugates (-61% modification) atconcentration 4 mg/ml (by Bluret assay) were incubated in theappropriate buffer. At given time intervals aliquots of these solutionswere injected into an HPLC equipped with Zorbax GF-450 column andRI-detector to quantitate mPEG-5000.

FIG. 2. Stability of SS-PEG and SC-PEG at 22° C.

Experimental Conditions

The activated PEG's were stored in the form of fine powder in tightlyclosed polypropylene containers. At given time intervals samples of eachpolymer were assayed for active acyl content.

FIG. 3. Reactivity of activated PEG's as a function of pH.

Experimental Conditions

To triethanolamine-borate buffer (0.3M, 1 ml) at the appropriate pH,stock solution of NAL in water (50 mM, 0.1 ml) was added followed bystock solution of the appropriate activated PEG in CH₃ CN (50 mM activeacyl, 0.1 ml). The resultant solution was vortexed and incubated at 28°C. for 1 hr. A mixture of the same components but leaving out SX-PEG wasused as a control. The TNBS--assay version of Snyder, et al. (1975)Anal. Biochem. 64, 284! was used to determine the unreacted NAL.

DESCRIPTION OF THE INVENTION

The present invention describes activated polyalkylene oxides having thegeneral structure: ##STR5## wherein R₁ is H--, H₃ C--, an oxycarbonylN-dicarboximide group, or any other functional group; wherein each R₂,R₃, and R₄ is an alkyl group which may be straight, branched,disubstituted, or unsubstituted, and wherein each R₂, R₃ and R₄ may beindependently the same as, or different from, the others of R₂, R₃, andR₄ ; wherein R₅ is an N-dicarboximide group; and wherein a is an integerbetween 1 and 1000 and each of b and c is an integer between 0 and 1000,and the sum of a, b, and c is between 10 and 1000.

More specifically, the invention relates to preparation and use of anew, activated PEG, namely, poly(ethylene glycol)-succinidyl carbonate(SC-PEG) and the bifunctional derivative of PEG, namely, polyethyleneglycol-bis-succinidyl carbonate (BSC-PEG). Furthermore,heterobifunctional derivatives of PEG are possible (Zalipsky and Barany,supra); one of the end groups is succinidyl carbonate and the other endgroup (R¹, see Scheme 2, infra) contains a different reactive functionalgroup, such as a free carboxyl group or an amine acid. These materialsreact readily with amino groups of proteins to afford PEG attachmentthrough stable urethane linkages (Scheme 2, below). The reactivity ofthe new agents, SC-PEG and BSC-PEG, are comparable to the conventionallyused SS-PEG. Thus, high degrees of modification are achievable in mildconditions (aqueous buffers, pH 5.8-11, preferably pH 7.0-9.5) withinabout 30-60 min. and moderate temperatures (4°-40° C). Additionally, theagents are soluble in a variety of organic solvents, thus being usefuland important in the coupling of low molecular weight, partiallyprotected peptides and other biologically useful ligands.

The PEG does not have to be of a particular molecular weight, but it ispreferred that the molecular weight be between 500 and 40,000; morepreferably between 2,000 and 20,000. The choice of molecular weight ofPEG is made based on the nature of the particular protein employed, forexample, the number of amino groups available for modification.

The N-hydroxysuccinimide released during protein modification isnon-toxic material that is often used in protein chemistry, especiallyfor preparation of biologically active protein-adducts. As in the caseof above mentioned carbonyldiimidazole activated PEG andPEG-phenylcarbonates, the product of protein modification using SC-PEGor BSC-PEG has PEG-chains grafted onto the polypeptide backbone throughcarbamate (urethane) linkages. However, due to the higher reactivity ofthe new agents, higher degrees of modification are achievable in shorterperiods of time. An additional advantage of succinimide carbonateactivated PEG is that those active functional groups that do not reactwith amino groups of a protein undergo fast aqueous hydrolysis producingN-hydroxysuccinimide, carbon dioxide and hydroxy-terminated PEG. This isof particular importance in the case of bifunctional PEG derivatives(BSC-PEG). These materials can serve a dual purpose: PEGylation andcrosslinking at the same time. The BSC-PEG, like any homobifunctionalmaterial can be used to crosslink two different proteins. When a BSC-PEGmolecule reacts with a protein via only one end group, the otherSC-group of the polymer, which does not react with the amine, ishydrolyzed and therefore no extraneous (potentially antigenic) residuesare introduced onto the PEG-protein conjugate.

Biological activities of proteins modified with SC-PEG and BSC-PEG arepreserved to a large extent as shown by the examples below. There is oneliterature precedent in which protein (tissue plasminogen activator)covalently conjugated with PEG via urethane links had a higher specificactivity than the same protein modified with SS-PEG to approximately thesame extent Berger & Pizzo, Blood, 71, 1641-1647, 1988!. ##STR6##

Naturally, the utility of SC-activated PEG-derivatives extends topreparation of PEG-conjugates of low molecular weight peptides and othermaterials that contain free amino groups.

A one-pot procedure for introduction of SC-groups onto PEG was developed(Scheme 3 below). First, polyethylene glycol chloroformate was generatedin situ by treatment of the polymer (PEG) with phosgene. The resultingchloroformate was then reacted with N-hydroxysuccinimide (HOSU) followedby triethylamine (TEA) to yield the desired activated derivatives ofPEG. The activated polymer preparations were purified from low molecularweight materials and determined to contain the theoretical amounts ofactive groups. ##STR7##

EXAMPLE 1

Preparation of SC-PEG: Methoxypolyethylene glycol of molecular weight5000 (Union Carbide, 60 g, 12 mmol) was dissolved intoluene/dichloromethane (3:1, 200 ml) and treated with a toluenesolution of phosgene (30 ml, 57 mmol) overnight. The solution wasevaporated to dryness and the remainder of phosgene was removed undervacuum. The residur was redissolved in toluene/dechloromethane (2:1, 150ml) and treated with solid N-hydroxysuccinimide (2.1 g, 18 mmol)followed by triethylamine (1.7 ml, 12 mmol). After 3 hours, thesolution-was filtered and evaporated to dryness. The residue wasdissolved in warm (50° C.) ethyl acetate (600 ml), filtered from traceinsolubles and cooled to facilitate precipitation of the polymer. Theproduct was collected by filtration and then recrystallized once morefrom ethylacetate. The product was dried in vacuo over P₂ O₅. The yieldwas 52.5 g (85% of theory).

To determine the active carbonate content of the product, samples of thepolymer were reacted with a measured amount of benzylamine indichloromethane and the excess of amine was titrated with perchloricacid in dioxane. These titrations indicated that 1 g of the productcontained 1.97×10⁻⁴ mole of active carbonate (101% of theoreticalcontent). I.R. (film on NaCl, cm⁻¹) characteristic bands at : 1812 and1789 (both C═O, succinimide); 1742 (C═O, carbonate); 1114 (CH₂ OCH₂). ¹³C--NMR (CDC1₃) : δ 168.5 (CH₂ C═O); 151.3 (O--CO₂); 71.9 (CH₃ OCH₂) 70.2(PEG); 68.7 (CH₂ CH₂ OCO₂); 68.0 (CH₂ CH₂ OCO₂); 58.9 (CH₃ O); 25.2 (CH₂C═O) ppm .

EXAMPLE 2

Preparation of BSC-PEG: Polyethylene glycol of molecular weight 4600(Union Carbide, 50g, 21.7 mequiv. OH) was converted to the correspondingbis-N-hydroxysuccinimide carbonate using a toluene solution of phosgene(50 ml, 96.5 mmol) and then N-hydroxysuccinimide (3.8 g, 23 mmol) andtriethylamine (3.2 ml, 23 mmol) following the procedure described inExample 1. After purification the product was obtained as a white powder(51 g, 96%). Active carbonate content was 4.0×10⁻⁴ mole/g (98% oftheoretical) as determined by titrations with benzylamine-perchloricacid. I.R. (film on NaCl, cm⁻¹) characteristic bands at : 1812 and 1789(both C═O, succinimide); 1742 (C↑O, carbonate); 1114 (CH₂ OCH₂). ¹³C--NMR(CDC1₃): δ 168.5 (CH₂ C═O); 151.3 (O--CO₂); 70. 2 (PEG); 68. 7(CH₂ CH₂ OCO₂); 68.0 (CH₂ CH₂ OCO₂); 25.2 (CH₂ C═O) ppm. H-NMR (CDC1₃) :δ 4.35 (m, 4H, CH₂ OCO₂); 3.55 (s, ˜400H, PEG); 2.74 (s, 8H, CH₂ C═O)ppm.

EXAMPLE 3

Preparation of polyethylene glycol-Bovine Serum Albumin conjugates(PEG-BSA):

A. SC-PEG (1 g) was added to a stirred solution of Bovine Serum Albumin(BSA) (100 mg) in 0.1M sodium phosphate, pH 7.8 (20 ml). Sodiumhydroxide (0.5N) was used to maintain pH 7.8 for 30 min. The excess offree PEG was removed by diafiltration using 50 mM phosphate bufferedsaline. The extent of modification of BSA was approximately 50% (30amino groups of BSA out of total 60 reactive with SC-PE) as determinedby trinitrobenzenesulfonate (TNBS) titration of amino groups Habeeb,Analyt. Biochem. 14, 328-336 (1966)!.

The same degree of modification was obtained when the experiment wasrepeated under identical conditions using SS-PEG instead of SC-PEG.

B. SC-PEG (1 g) was added to a stirred solution of BSA (100 mg) in 0.1Msodium borate, pH 9.2. Sodium hydroxide (0.5N) was used to maintain pH9.2 for 30 min. The excess of free PEG was removed by diafiltration andthe product assayed for the number of free amino groups. Approximately68% (41) of the amino groups of the native BSA were modified.

C. BSC-PEG (1 g) was added to a stirred solution of BSA (100 mg) in 0.1Msodium borate, pH 9.2. Sodium hydroxide (0.5N) was used to maintain pH9.2 for 30 min. The excess of free PEG was removed by diafiltration andthe product assayed for the number of free amino groups. Approximately80% (48) of the amino groups of the native BSA were modified. Analysisof the product by HPLC (Gel Filtration) indicated that over 65% ofPEG-BSA was in intermolecularly crosslinked form and about 35% of theproduct had the same molecular weight as PEG-BSA from Example 3B.

EXAMPLE 4

Preparation of PEG-glutaminase: A solution of glutaminase Pseudomonas 7A(200 mg) in 0.1M sodium phosphate, pH 7.8 was treated with SC-PEG (4.7)g). The solution was stirred and pH 7.8 was maintained for 30 minutes.The excess free PEG was removed by diafiltration using 50 mM PBS. Theextent of modification of glutaminase was 74% as determined bytrinitrobenzenesulfonate titration of amino groups (see Habeeb, 1966above). The PEG-glutaminase product preserved 81% of the enzymaticactivity of the parent glutaminase.

EXAMPLE 5

Preparation of PEG-Trypsin: A solution of bovine pancreatic trypsin(Boeringer-Mannheim, 120 mg in 20 ml), that was dialyzed overnight at 4°C. against 20 mM CaC1₂ in 1 mM HCl, was brought to 25°C. and treatedwith SC-PEG (600 mg) for 30 min. During this time pH 7.8 was maintainedin the reaction vessel by automatic titration with 0.5N NaOH. Thesolution was acidified to pH 3 and extensively diafiltered to removeexcess of free polymer using 20 mM CaC1₂ in 1 mM HCl as a replacementfluid. The modified trypsin had approximately half of the free aminogroups of the parent enzyme (7 PEG chains per trypsin molecule) asdetermined by TNBS titration of amino groups (Habeeb 1966). ThePEG-trypsin product preserved 96% of enzymatic activity of the parentenzyme towards Nα-benzoyl-L-arginine ethyl ester.

EXAMPLE 6

Preparation of PEG-Arginase: A solution of bovine liver arginase (Sigma,90 mg) in 0.1M NaCl (20 ml) was treated with SC-PEG (1.3 g) at 27°C.while pH 8.0 was maintained by automatic titration with 0.5N NaOH. After30 min. the reaction mixture was diafiltered using 50 mM PBS as areplacement fluid. Approximately 64% of the amino groups of the nativearginase were modified (56 PEG chains per arginase molecule). ThePEG-arginase product retained 70% of specific activity of the nativeenzyme when assayed with 2,3-butanedione (BUN-urea reagent) at pH 9.5.

EXAMPLE 7

Other polypeptides, including chymotrypsin, asparaginase, and adenosinedeaminase, have been modified with SC-PEG using the procedures set forthherein.

The methods of using SC- and/or BSC-functionalized polyalkylene oxides,such as PEG and its copolymers are generally applicable to thepreparation of other modified polypeptides and other biologically activecomponents having amino groups.

While the present invention has been described by reference toN-hydroxysuccinimide derivatives, it will be obvious to those skilled inthe art that other N-hydroxydicarboximides may be substituted therefor.Typical of such derivatives are N-hydroxyphthalimide,N-hydroxyglutarimide, N-hydroxytetrahydrophthalimide,N-hydroxy-5-norbornene-2,3-dicarboximide or other N- disubstitutedderivatives of hydroxylamine.

EXAMPLE 8

Comparison of SC-PEG to SS-PEG

A. As seen in Scheme 2, the product of protein modification using SC-PEGhas PEG-chains grafted onto the polypeptide backbone through carbamate(urethane) linkages. Greater stability of the urethane linkage relativeto the ester bond produced upon use of SS-PEG (see Scheme 1) wasexpected to prove a key difference between the two activated PEG's andthe corresponding PEG-protein conjugates. Our studies indeed confirmedthis expectation. FIG. 1 shows the results of GF-HPLC measurements ofthe amounts of free mPEG produced as a result of incubation of PEG-BSAconjugates derived from each of the activated PEG's. Considerably higherstability of the SC-PEG-derived conjugate is apparent.

B. To estimate the reactivities of SC-PEG and SS-PEG, kineticmeasurements of hydrolysis of the activated polymers in phosphate bufferand their aminolysis by Nα-acetyl-lysine (NAL) were performed. Theresults of these experiments are summarized in Table 1. It is clear fromthese data that SS-PEG is a more reactive reagent than SC-PEG. Thedifference in hydrolysis rates was larger than the difference inaminolysis rates; consequently, SC-PEG showed more favorable K_(am)/K_(h) ratios. The slower hydrolysis of SC-PEG was also manifested insuperior storage stability of the reagent (FIG. 2).

C. Reactivity of the activated PEGs as a function of pH was determinedusing NAL as a model for the ε-amino group of a protein. Each of theactivated PEGs was reacted with an equimolar amount of NAL at differentpH's, and measured the unreacted NAL using the TNBS-assay (FIG. 3). Theoptimal pH for use of SC-PEG was found to be about 9.3. It is notadvisable to use SS-PEG at pH>8.0, due to the limited stability ofPEG-succinate ester. However, even at pH values less than 8.0 thisactivated PEG was found to be very reactive.

D. Both reagents showed high reactivity towards Trypsin yieldingcomparably modified enzyme derivatives in mild conditions (pH 7.5-8.5)within 30 min. The products were purified by diafiltration, and thedegrees of modification were determined by fluorometric assay, accordingto Stocks, et al. (1986) Anal. Biochem. 154, 232.

All PEG-modified trypsin derivatives were essentially lacking (<1% ofnative) proteolytic activity as determined by the Azocoll assay Chavira,et al. (1984) Anal. Biochem. 136, 446!. The specific activities ofrepresentative SS-and SC-PEG modified trypsins towards low molecularweight substrates are summarized in Table 2. The modifications producedhardly any changes in esterolytic activities towards benzoyl-L-arginineethyl ester, but did enhance the activities towards p-nitroanilides.Michaelis-Menten kinetic constants for several SC- and SS-PEG modifiedtrypsins were measured using ZAPA as the substrate. These results,summarized in Table 3, indicate that, while V_(max), K_(cat) and K_(cat)/K_(m) were increasing gradually with the extent of modificaitons, K_(m)values were decreasing.

As compared to SS-PEG, SC-PEG is a less reactive yet more. selectivereagent. This is evidenced by its higher K_(am) /K_(h) ratios and betterstorage stability. SC-PEG is a sufficiently reactive reagent to producePEG-protein conjugates under mild conditions within 30 min. SC-PEG canbe used in a broader pH range than SS-PEG, showing the highestreactivity at pH═9.3. PEG-protein conjugates obtaining through use ofSC-PEG are chemically more stable than SS-PEG derived conjugates. ThePEG-Trypsin conjugates produced by both activated PEG's have verysimilar properties: They show no proteolytic activity, well preservedesterolytic activity, and dramatically increased activity towardsp-nitroanilide substrates. Michaelis-Menten constants of the modifiedenzymes indicate that the attachment of PEG to trypsin causes anincrease in both the rate of turnover of ZAPA and its affinity towardsthe modified enzymes.

                  TABLE 1                                                         ______________________________________                                        Comparison of first order rate constants for hydrolysis (K.sub.h) and         aminolysis (K.sub.am) of SC-PEG and SS-PEG.sup.a                                       Hydrolysis: K.sub.h.sup.b (min.sup.-1) ×                                                Aminolysis: K.sub.am.sup.c (min.sup.-1) ×      Temp.    10.sup.3 and  t.sub.1/2 (min)!                                                                10.sup.3 and  K.sub.am /K.sub.h !                    pH   (°C.)                                                                          SC-PEG    SS-PEG  SC-PEG  SS-PEG                                 ______________________________________                                        7.0  4       0.87  793!                                                                              1.84  376!                                                                            2.64  3.0!                                                                            3.74  2.0!                                  27      6.05  115!                                                                              10.4  67!                                                                             26.4  4.4!                                                                            41.4  4.0!                                  37      14.2  49! 25.9  27!                                                                             81.7  5.8!                                                                            104  4.0!                              7.4  22      5.37  129!                                                                              10.7  65!                                                                             29.1  5.4!                                                                            42.7  4.0!                                  27      9.0  77!  16.0  43!                                                                             48.6  5.4!                                                                            73.6  4.6!                                  37      19.3  36! 37.6  18!                                                                             145  7.5!                                                                             193  5.1!                              7.8  4       1.37  505!                                                                              2.58  268!                                                                            12.4  9.1!                                                                            15.0  5.8!                                  27      10.3  67! 21.6  32!                                                                             130  12.6!                                                                            152  7.0!                                   37      21.8  32! 48.8  14!                                                                             226  10.6!                                                                            267  5.5!                              ______________________________________                                         .sup.a All the measurements were performed by following the appearance of     Nhydroxysuccinimide anion (--OSu) at 260 nm in 0.008M sodium phosphate;       concentration of PEGbound succinimidyl active acyl at time zero                SXPEG!.sub.0 was 0.1 mM; in aminolysis experiments concentration of          N.sup.αacetyl-lysine at time zero  NAL!.sub.0 was 3 mM.                 .sup.b K.sub.h = Rate.sub.h / SXPEG!.sub.0, where Rate.sub.h = dA.sub.260     /dt × 1 E.sub.260 × 1F; ε.sub.260 = 8500M.sup.-1          cm.sup.-1 is an extintion coeficient of --OSu; and F =  --OSu!/( HOSu! +       --OSu)! = (1 + 10.sup.6.0 pH).sup.-1.                                        .sup.c K.sub.am = Total Rate/ SXPEG!.sub.0 - K.sub.h. The Total Rate in       aminolysis experiments was calculated the same way as Rate.sub.h in           hydrolysis experiments.                                                  

                                      TABLE 2                                     __________________________________________________________________________    SUMMARY OF MODIFICAITON, ESTEROLYTIC ACTIVITY, AND AMIDOLYTIC                 ACTIVITY DATA FOR TYPSIN AND ITS mPEG DERIVATIVES                             Trypsin  Modif.sup.b                                                                       BAEE.sup.c                                                                        %    BAPA.sup.d                                                                        %   ZAPA.sup.d                                                                         %                                          Derivatives.sup.a                                                                      (%) (μ/mg)                                                                         Native                                                                             (μ/mg)                                                                         Native                                                                            (μ/mg)                                                                          Native                                     __________________________________________________________________________    Native Trypsin                                                                         0   92.4                                                                              100  1.26                                                                              100 7.81 100                                        SC-PEG.sub.N -Trypsin                                                         N = 6    42.3                                                                              103 112  2.26                                                                              179 15.3 196                                        N = 7    45.8                                                                              87.9                                                                              95.1 2.38                                                                              188 17.5 224                                        N = 9    58.8                                                                              90.1                                                                              97.5 2.67                                                                              212 18.9 242                                        N = 12   77.9                                                                              85.1                                                                              92.2 3.83                                                                              304 25.5 326                                        SS-PEG.sub.N -Trypsin                                                         N = 7    44.8                                                                              102 110  3.25                                                                              258 18.8 241                                        N = 12   770 94.3                                                                              102  4.34                                                                              344 24.7 316                                        __________________________________________________________________________     .sup.a For SXPEG.sub.hTypsin, N = 15 × (% Modif)/100 and is rounded     to the nearest integer.                                                       .sup.b The percent of amino groups modified was determined by the             fluorescamine assay  Stocks, et al. (1986) Anal. Biochem. 154, 232            .sup.c The BAEE (N.sup.αbenzoyl-L-arginine ethyl ester) trypsin         assay was done at pH 7.8, 37° C. w/ a substrate conc'n of 0.5 mM.      The extinction coefficient was ε.sub.253 = 808M.sup.-1 cm.sup.-1       Kezdy, et al. (1965) Biochemistry 4, 99!.                                    .sup.d The BAPA (N.sup.αbenzoyl-D, γ Larginine-p-nitroanilide     and ZAPA (N.sup.αCBZ-L-arginine-p-nitroanilide) amidolytic assays       were done w/ a substrate conc'n of 1 mM in 50 mM TrisHCl pH 8.1, 10 mM        CaCl.sub.2, at 37° C. The extinction coefficient for pnitroaniline     ε.sub.410 = 8800M.sup.-1 cm.sup.-1, was used in both assays.     

                  TABLE 3                                                         ______________________________________                                        MICHAELIS-MENTEN CONSTANTS FOR THE AMIDOLYTIC                                 ACTIVITY OF NATIVE TRYPSIN AND ITS mPEG DERIVATIVES.sup.a                                 Km     V.sub.max K.sub.cat                                                                           Kcat/Km                                    Trypsin Derivatives                                                                       (mM)   (μM/min)                                                                             (min.sup.-1)                                                                        (mM.sup.-1 · min.sup.-1)          ______________________________________                                        Native Trypsin                                                                            1.08   15.7      378    349                                       SC-PEG.sub.N -Trypsin                                                         N = 7       0.29   19.6      470   1626                                       N = 9       0.21   20.2      484   2290                                       N = 12      0.11   22.9      549   4973                                       SS-PEG.sub.h -Trypsin                                                         N = 7       0.21   18.6      447   2172                                       N = 12      0.13   22.5      539   4159                                       ______________________________________                                         .sup.a The measurements took place at 37° C. with a constant           trypsin protein concentration of 1.0 μg/ml (via Bluret assay).             Ncarbobenzoxy-L-arginine-p-nitroanilide (ZAP) was used as a substrate in      concentrations varying from 0.02 to 1.71 mM in 50 mM TrisHCl pH 7.8, 10 m     calcium chloride. The constants were calculated from LineweaverBurk plots     of the initial rates of the appearance of pnitroaniline (ε.sub.41     = 8800M.sup.-1 cm.sup.-1).                                               

What is claimed is:
 1. A process for preparing a polyalkylene oxidehaving a terminal oxycarbonyl-oxy-N-dicarboximide group, whichcomprisesa) treating a polyalkylene oxide with phosgene to form apolyalkylene oxide chloroformate in situ: b) reacting said polyalkyleneoxide chloroformate with an N-hydroxy-N-dicarboximide followed by a baseto form a polyalkylene oxide having a terminaloxycarbonyl-oxy-N-dicarboximide group.
 2. The process of claim 1,wherein the N-hydroxy-N-dicarboximide is N-hydroxysuccinimide.
 3. Theprocess according to claim 1 wherein the base is triethylamine.
 4. Theprocess of claim 1 wherein said polyalkene oxide having saidoxycarbonyl-oxy-N-dicarboximide has the structure:

    R.sub.1 --(O--R.sub.2).sub.a --(O--R.sub.3).sub.b --(O--R.sub.4).sub.c --O--CO--O--R.sub.5

wherein R₁ is H--, H₃ C--or a carbonyl-oxy-N-dicarboximide group; R₂,R₃, and R₄ are independently selected from the group consisting ofstraight and branched alkyl groups; R₅ is an N-dicarboximide group; andα is and integer between 1 and 1,000 and b and c are independently zeroor an integer between 1 and 1,000.
 5. The process of claim 4 wherein R₂,R₃, and R₄ are independently selected from the group consisting of --CH₂--CH₂ --, --CH₂ --CH(CH₃)--, and --CH₂ CH₂ CH₂ CH₂ --.
 6. The process ofclaim 4 wherein R₁ and R₅ are carbonyl-oxy-N-dicarboximide groups andthe N-dicarboximide of R₁ and R₅ are independently selected from thegroup consisting of N-succinimide, N-phthalide, N-glutarimide,N-tetrahydrophthalimide and N-norborene-2,3-dicarboximide groups.
 7. Theprocess of claim 4 wherein R₅ is an N-succinimide group.
 8. The processof claim 4 wherein said polyalkene oxide having saidoxycarbonyl-oxy-N-dicarboximide has the structure: ##STR8## wherein R₁is H₃ C-- or H-- and (α) is an integer between 1 and 1,000.
 9. Theprocess of claim 4 wherein said polyalkene oxide having saidoxycarbonyl-oxy-N-dicarboximide has the structure: ##STR9## wherein (α)is an integer between 1 and 1,000.
 10. The process of claim 1, whereinsaid polyalkylene oxide has a molecular weight from about 500 to about40,000.
 11. The process of claim 10, wherein said polyalkylene oxide hasa molecular weight from about 2,000 to about 20,000.
 12. The process ofclaim 11, wherein said polyalkene oxide has a molecular weight about5,000.
 13. The process of claim 1, wherein said polyalkylene oxidehaving said terminal oxycarbonyl-oxy-N-dicarboximide group ispolyethylene glycol-succinidyl carbonate.
 14. The process of claim 1,wherein said polyalkylene oxide having said terminaloxycarbonyl-oxy-N-dicarboximide group is polyethyleneglycol-bis-succinidyl carbonate.